The document discusses the significance of DNA-protein interactions in gene regulation and genome stability, detailing various techniques like 3C, 4C, 5C, Hi-C, and ChIA-PET that study these interactions. It emphasizes the advancements in studying the three-dimensional genome organization and how these methods can analyze chromatin interactions at high resolution. Additionally, it outlines the methodologies and limitations of each technique in understanding DNA topology and gene regulation.

1. By: Kashvi Jadia(MSc Biotechnology-sem-9)
Professor: Dr. Anjali Soni
Department of Biotechnology, VNSGU.
DNA PROTEIN INTERACTION

2. INTRODUCTION
➔It is well known that the chromosomes are organized in the
nucleus and this spatial arrangement of genome play a crucial
role in gene regulation and genome stability.
➔DNA- Protein interactions play very vital role in any living cell.
➔It controls replication, transcription, recombination, DNA
repair etc.
➔There are several types of proteins found in a cell.But only
those proteins interact with DNA which have the DNA
binding domains.

3. ➔Each DNA binding domain has at least one motif which is a
conserved amino acid sequence of this protein which can
potentially recognize a double stranded or a single stranded
DNA.
➔There are mainly two broad types of DNA protein interactions.
➔ Sequence specific DNA binding:
➔ Sequence non-specific DNA binding:

4. 1. Sequence specific DNA binding:
➔A DNA binding protein binds to a DNA on a site having
specific nucleotide sequence
➔Frequently involve DNA major groove.
➔Interaction maintained by Hydrogen bonds, Ionic
Interaction , Van der waals forces.
1. Sequence non-specific DNA binding:
➔The DNA binding protein can bind to a DNA in a random
position on the DNA.
➔In replication

5. ➔ Traditionally, nuclear organization is studied by microscopy, and
thus it is appropriate to start by highlighting some important
observations made under the microscope.
➔ The segregation of active and inactive chromatin inside the
nucleus raises the possibility that nuclear positioning affects gene
activity. This idea is supported by DNA fluorescence in situ
hybridization (FISH) observations that certain genes (e.g., HoxB and
uPA) loop out of their chromosome territory upon activation.

6. ➔ Some studies show inactive genes located in the interior of CTs
(chromosome territory)and active genes concentrated at the
territory periphery , but active genes can be transcribed from
inside of CTs.
➔ Moreover, regions with a high density of coordinately
expressed genes locate in loops that extend outside of CTs in
expressing cells but not in non expressing cells.
➔ Localization outside of CTs also occurs at genomic regions with
a high-density of broadly expressed genes.
➔ Hence, it has been suggested that there is a correlation
between high levels of transcriptional activity and localization
outside of CTs.

7. ➔ The power of FISH and other microscopy methods lies in their
ability to do single-cell analyses of gene positioning.
➔ However, on a genomic and cell population scale, they are
limited in throughput and resolution.
➔ It is therefore unclear whether they uncover general principles of
nuclear organization or the peculiarities of individual genes.

8. ➔ Ten years ago, Dekker et al. (2002) developed 3C technology, a
biochemical strategy to analyze contact frequencies between
selected genomic sites in cell populations. Since then, various 3C-
derived genomics methods have been developed.
➔ In comparison with microscopy, 3C-based methods enable
more systematic DNA topology studies at a higher resolution.

9. ➔ Advantage: These technologies can put observations made on
single genes in selected cells in the context of genomic behavior
in cell populations. The generated DNA contact maps start
teaching us the rules that dictate genome structure and
functioning inside the cell.
➔Morden techniques:
◆ 3C (Chromosome conformation capture)
◆ 4C (Chromosome conformation capture on chip)
◆ 5C (Chromosome conformation capture carbon copy)
◆ HiC (High-throughput Chromosome Conformation Capture)
◆ ChlAPET (Chromatin interaction analysis by paired-end tag
sequencing)

11. 3C (Chromosome Conformation Capture):
➔ Chromosome folding is modulated as cells progress through the
cell cycle. During mitosis, condensins fold chromosomes into
helical loop arrays. In interphase, the cohesin complex generates
loops and topologically associating domains (TADs), while a
separate process of compartmentalization drives segregation of
active and inactive chromatin.
➔The term "3C DNA-protein interaction" refers to a laboratory
technique used to study the three-dimensional (3D) interactions
between DNA and proteins within the nucleus of a cell.

12. ➔ The strategy of 3C to discover genomic architecture is based on
quantifying the frequencies of contacts between distal DNA
segments in cell populations.
➔The 3C technique allows researchers to investigate how different
regions of the genome physically interact with each other and with
specific proteins.
➔This helps to understand the higher-order chromatin structure and
how it influences gene regulation and other genomic processes.

13. ➔The 3C technique involves several key steps:
1. Cross-Linking:
2. Cell Lysis and Restriction Enzyme
Digestion:
3. DNA Ligation:
4. Reverse Cross-Linking and Purification:
5. Quantitative Analysis:

14. 1. Cross-Linking:
➔ The initial step in 3C and 3C-derived methods is to establish a
representation of the 3D organization of the DNA. To this end, the
chromatin is fixed using a fixative agent, most often formaldehyde.
1. Cell Lysis and treatment with Restriction Enzyme:
➔ Next, the fixed chromatin is cut with a restriction enzyme
recognizing 6 base pairs (bp)—such as HindIII, BglII, SacI, BamHI,
or EcoRI—or with more frequent cutters, such as AciI or DpnII.

16. 3. DNA Ligation:
➔ In the subsequent step, the sticky ends of the cross-linked DNA
fragments are religated under diluted conditions to promote
intramolecular ligations (i.e., between cross-linked fragments).
4. Reverse crosslinking and purification:
➔ DNA fragments that are far away on the linear template, but
colocalize in space(by reverse crosslinking), can, in this way, be
ligated to each other.
➔ The ligation mixture is purified. 3C yields a genome-wide ligation
product library in which each ligation product corresponds to a
specific interaction between the two corresponding loci.

17. 4. Quantitative Analysis:
➔ A template is thereby created that is, in effect, a one-
dimensional (1D) cast of the 3D nuclear structure.
➔ Conventional 3C uses polymerase chain reaction (PCR) with
specific primers to detect 3C ligation products one at a time.
The PCR primers are designed to anneal 100–150 bp
upstream of and downstream from the newly formed restriction
site of the ligation product

18. Limitation:
➔ In order to appreciate loops visualized by 3C-based
technologies, one needs to find the anchor interacting with a
distant sequence more frequently than with intervening sequences.
Therefore, 3C methods intrinsically rely on quantitative rather than
qualitative measurements.
➔ The importance of this assessment is underscored by the
following consideration:
➔ At most alleles, cross-linking will result in larger chromatin
aggregates with many DNA fragments together (“hairballs”), within
which all DNA ends compete with each other for ligation to the
anchor fragment.

19. 4C (Circular Chromosome Conformation Capture):
➔4C extends the 3C method by allowing the investigation of
interactions between a specific genomic region of interest
(viewpoint) and the entire genome.
➔In this method, DNA-protein complexes are crosslinked using
formaldehyde. The sample is fragmented, and the DNA is ligated
but with a modification that includes a biotinylated primer specific
to the viewpoint region and digested..
➔Use the biotinylated primer to perform a second round of
PCR(inversed PCR) with primers targeting the ligated DNA
fragments.
➔ Analyze the PCR products by quantitative PCR or high-throughput
sequencing to identify interacting regions with the viewpoint region.

21. ❑Advantages:
1. Preferred strategy to assess the DNA contact profile
of individual genomic sites
2. Highly reproducible data
❑Disadvantages:
1. Will miss local interactions (< 50 kb) from the region
of interest
2. Large circles do not amplify efficiently

22. 5C :
➔ In 5C , the 3C template is hybridized to a mix of oligonucleotides, each
of which partially overlaps a different restriction site in the genomic
region of interest.
➔ Pairs of oligonucleotides that correspond to interacting fragments are
juxtaposed on the 3C template and can be ligated together. Since all
5C oligos carry one of two universal sequences at their 5′ ends, all
ligation products can subsequently be amplified simultaneously in a
multiplex PCR reaction.

23. ➔ Readout of these junctions occurs either on a microarray or by
high-throughput sequencing.Restriction fragments of interest are
selected throughout the genome, and a 5C primer is designed for
each of them.
➔ 5C uses two types of primers - forward primers and reverse
primers.
➔ Either a forward or a reverse primer is designed for each restriction
fragment. These primers are designed so that forward and
reverse primers anneal across ligated junctions of head-to-
head ligation products present in the 3C library.

24. ➔ 5C primers that are annealed next to each other are then ligated
with Taq ligase. This step generates a 5C library, which is amplified
with universal PCR primers that anneal to the tails of the 5C
primers.
➔ Forward and reverse 5C primers are only ligated when both are
annealed to a specific 3C ligation product. Therefore, the 3C
library determines which 5C ligation products are generated and
how frequently. As a result, the 5C library is a quantitative “carbon
copy” of a part of the 3C library, as determined by the collection of
5C primers.

25. ⚫ (A) Primers used for PCR detection
of 3C ligation product are designed
so that they anneal to the same
strand of genomic DNA and are able
to prime amplification of a head-to-
head 3C ligation product.
⚫ (B) Primers used for 5C detection
are designed so that they anneal to
the opposite strands of genomic
DNA and are able to detect a head-
to-head 3C ligation product.
Arrowheads on primers indicate the
3′ ends. The non-annealing gray
sections of the 5C primers represent
the universal tails (see the main
text). Forward primers have a 5′
universal tail, whereas reverse
primers carry a universal tail at their
3′ ends.

26. ➔5C was developed and validated by analyzing the human β-
globin Locus and a conserved gene desert region located
on human chromosome 16.
➔5C analysis also identified a looping interaction between the β-
globin Locus Control Region (LCR) and the γ–δ intergenic
region.

27. Hi-C (High-throughput Chromosome Conformation
Capture):
➔Hi-C is an advanced 3C-based method that allows genome-wide
analysis of chromatin interactions, providing a comprehensive
view of chromosomal interactions at a high resolution.
➔Similar to the classic 3C technique, Hi-C measures the
frequency (as an average over a cell population) at which two
DNA fragments physically associate in 3D space, linking
chromosomal structure directly to the genomic sequence.
➔ The general procedure of Hi-C involves generation of segments
as we did in 3C.

28. ➔After restriction enzyme digestion, the sticky ends are filled in with
biotin-labeled nucleotides followed by blunt-end ligation.
➔As a result, biotin-marked ligation junctions can be purified more
efficiently by streptavidin-coated magnetic beads, and chromatin
interaction data can be obtained by direct sequencing of the Hi-C
library.
➔only biotinylated junctions are selected for further high-throughput
sequencing and computational analysis
➔While 3C focuses on the analysis of a set of predetermined genomic
loci to offer “one-versus-some” investigations of the conformation of
the chromosome regions of interest, Hi-C enables “all-versus-all”
interaction profiling by labeling all fragmented chromatin with a
biotinylated nucleotide before ligation.

30. ➔Analyses of Hi-C data not only reveal the overall genomic
structure of mammalian chromosomes but also offer insights into
the biophysical properties of chromatin as well as more specific,
long-range contacts between distant genomic elements (e.g.
between genes and regulatory elements).
➔In recent years, Hi-C has found its application in a wide variety of
biological fields, including cell growth and division,transcription
regulation, fate determination development, disease, and genome
evolution.
➔By combining Hi-C data with other datasets such as genome-wide
maps of chromatin modifications and gene expression profiles, the
functional roles of chromatin conformation in genome regulation
and stability can also be delineated.

31. ChIA-PET:
➔ The cross-linked chromatin interaction nodes bound by protein
factors are enriched by ChIP
➔ Remote DNA elements tethered together in close spatial distance in
these chromatin interaction nodes
➔ They are connected through proximity ligation with oligonucleotide
DNA linkers.
➔ Exmaple(MmeI):
➔ linker sequences are created such a way that they not only contain
MmeI restriction sites for PET extraction, but also include specific
nucleotide barcodes

33. ➔ Upon MmeI digestion, the resulting PET construct contains a 20 bp
head tag, a 38 bp linker sequence, and a 20 bp tail tag, which is
the template for next generation paired-end sequencing, for
example, Illumina paired-end sequencing .
➔ When PETs are mapped to the corresponding reference genome
sequences, the genomic distance between the two mapped tags
will reveal whether a PET is derived from a self-ligation product of
a single DNA fragment (short genomic distance) or an inter-
ligation product of two DNA fragments (long genomic distance,
or inter-chromosomal)

36. Reference:
1. https://molecularcytogenetics.biomedcentral.com/articles/10.1186/s13039-018-
0368-2
2. https://cshprotocols.cshlp.org/content/2018/9/pdb.top097832.full#:~:text=Outline%2
0of%20the%203C%20Technique,identity%20of%20the%20interacting%20loci.
3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3258961/
4. https://pure.eur.nl/ws/files/46745398/Simonis07_3C-5C_nuclear_organization.pdf
5. https://cmb.i-learn.unito.it/mod/wiki/prettyview.php?pageid=116
6. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2872882/